Proteases are the major participants in the proteolysis in human body, and can be classified into serine proteases, cysteine proteases, aspartyl proteases, threonine proteases, metalloproteinases, etc. depending on proteolytic mechanism. Current studies mainly focus on cysteine proteases wherein papain-like cysteine proteases are the largest subfamily. Papain-like cysteine proteases in mammals belong to cathepsins. Cathepsins, most members of which are present in lysosomes, can be activated in an acidic environment, and include most of the cysteine proteases, and a small number of aspartic proteases (Cathepsin D, E) and serine proteases (Cathepsin A, G). Depending on substrate specificity, cathepsins can also be classified into endopeptidases (Cathepsin B, F, H, K, L, S, V), exopeptidases (Cathepsin B, C, H, X), amino-peptidases (Cathepsin C, H) and carboxypeptidases (Cathepsin B, X). Cathepsins in human body mainly include Cathepsin B, C, F, H, K, L, O, S, V, W and X, which are closely related to a variety of physiological and pathological processes in human body. Cathepsins are highly conservative in sequence, and their steric structure consists of two domains (i.e. L (Left) domain and R (Right) domain) that are almost the same size, and between the two domains, there is a V-shaped active site cleft, in which the active residues, such as Cys25 in the L domain and His159 and Asn175 in the R domain, are exposed.
Cathepsin B, which is one of the most studied enzyme among all cysteine proteases, is widely present in a variety of tissues in mammals, and is the first lysosomal protease that was found to be associated with breast cancer. In addition, Cathepsin B is also closely associated with the development and progression of a variety of human tumors. Tumor cells can secrete Cathepsin B. This secretory Cathepsin B cannot be uptaken by lysosomes due to the lack of a recognition marker for mannose-6-phosphate receptor, and therefore is generally present in the form of proenzyme in endochylema and extracellular region. Since tumor cells can acidify their surrounding environment, they can activate the proenzyme to form active Cathepsin B. The activated Cathepsin B can not only participate in the degradation of extracellular matrix components, but also activate the proteolysis cascade reaction, finally resulting in the generation of a substance capable of degrading a variety of extracellular matrix components. In addition, as compared to Cathepsin B secreted by normal cells, the activity of Cathepsin B secreted by tumor cells is not influenced, and is even enhanced in neutral and alkaline environments. Therefore, the expression level and/or activity of Cathepsin B was found to be increased in a variety of human and animal tumors such as gastric cancer, bladder cancer, colon cancer, neuroglioma, and melanoma. It was reported that in the tissues of malignant tumor such as cervical cancer, lung cancer, breast cancer, and prostate cancer, the expression and activity of Cathepsin B was 2 times or even 3 to 9 times higher than that of the adjacent normal tissues, and it was believed that an increase in the activity and concentration of Cathepsin B was a risk factor for tumor invasion, metastasis, and poor prognosis in patients with transitional cell carcinoma.
Normal bone metabolism in human body depends on the dynamic balance between bone formation and bone resorption. Osteoporosis relates to a metabolic imbalance between bone resorption and bone formation, and is a disease developed when bone resorption exceeds bone formation. Osteoporosis is a common disease or a frequently-occurring disease found in old people, especially menopausal and postmenopausal women. During bone resorption, osteoclasts are first attached to the bone surface so as to form a relatively sealed microenvironment for bone resorption, secrete protons and proteases, dissolve bone minerals, and then degrade bone matrix, resulting in bone void formation. There are two major enzymes involved in the degradation of bone matrix during osteolysis, i.e. cysteine proteases and matrix metalloproteinases, and among cysteine proteases, Cathepsin K plays a major role. Cathepsin K is selectively and abundantly expressed in osteoclasts, and its physiological substrate is the type I collagen with a content of 95% in the organic bone matrix. Furthermore, Cathepsin K can also degrade osteopontin and osteonectin in bone matrix, and is a cysteine protease that is expressed in the highest level in osteoclasts and has the strongest osteolytic activity, and its ability of degrading bone collagen is much higher than that of other matrix metalloproteinases. Cathepsin K is a key enzyme during bone resorption, and is also a hotspot in osteoporosis research in recent years. In addition to Cathepsin K, Cathepsin L is also present in the resorption microenvironment formed by osteoclasts, and has a strong collagen-dissolving ability and is also directly involved in the degradation of bone matrix. In addition, it has been demonstrated in studies that Cathepsin B and S exhibit bone-resorption ability in vive and in vitro, and are also associated with the development of osteoporosis.
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by inflammation of synovial membrane of joint. Continuous recurrence of synovitis can lead to destruction of cartilage and bone in joint, joint dysfunction, and even disability. Osteoarthritis (OA) is a common disease causing joint pain, resulted from non-inflammatory lesions in movable joints due to the degeneration of articular cartilage and the formation of new bone on the surface and edge of joint. Cartilage contains two main components, one of which is type II collagen that can form a 3-D fiber network structure to make the tissue extensible, and the other of which is aggrecan that ensures the tenacity of cartilage, excessive degradation of either of the two protein components will lead to the cartilage destruction in joint. It has been found in studies that the degradation of type II collagen was only associated with matrix metalloproteinase (secreted by chondrocyte) and Cathepsin K. The expression level of Cathepsin K was increased in the joints of RA and OA patients, and Cathepsin K could be further activated by inflammatory cytokines, indicating that Cathepsin K may be the main participant in cartilage destruction in RA and OA. In addition, it has also been found in many experiments that Cathepsin B and Cathepsin L were expressed in high levels in synovial membrane and synovial fluid of joints in RA patients, all the cathepsins appeared in the inflammatory joint could hydrolyze aggrecan, and Cathepsin B could also play an indirect role by participating in the proteolytic cascade reaction in the joint and activating matrix metalloproteinase precursor.
It is found in the current researches that Ebola virus infection is closely associated with the type I membrane-fusion protein GP encoded by a viral gene. The membrane-fusion protein GP consists of two subunits, GP1 and GP2. An Ebola virus particle has an enveloped helical nucleocapsid. An enveloped virus cannot replicate its pathogenic gene unless the envelope protein is fused with a host cell membrane or endosomal membrane. When an Ebola virus invades a cell, upon the attachment of the virus to the cell surface, its envelope-fusion protein GP has to be bound to Cathepsin B or L to alter its conformation so that it can be fused with the host endosomal membrane and then has the carried genetic material injected into the cell, resulting in massive replication in the infected cell. Bale et al. have shown in their studies that the Ebola virus GP1 and GP2 proteins cannot accomplish conformational alteration and mediate fusion on their own in acidic conditions, and need to be bound to other cellular proteins so as to accomplish conformational alteration. In the article published by Kathryn Schornberg et al., it is demonstrated by the experiments using chemical inhibitors and small interfering RNAs that Cathepsin B and Cathepsin L play a role in Ebola virus glycoprotein-mediated infection. It is demonstrated in the study that Cathepsin B or L finally cleaved GP1 into a 19-Kd fragment. However, it is not clear yet as to how the fragment triggers enzyme fusion and induces fusion. Matthew Brecher et al. found by binding different forms of GPs to liposomes that when a 19-Kd fragment was fused with a liposome, the activation temperature was the lowest, indicating that it was the optimal form for fusion.
Cathepsin L can promote the invasion and metastasis of tumor by catalyzing the degradation of matrix membrane. Cathepsin L has been found to be highly expressed in various cancers such as kidney cancer, testicular cancer, breast cancer, ovarian cancer, colon cancer, bladder cancer and thyroid cancer. Cathepsin S also plays an important role in the pathogenesis of degenerative diseases and autoimmune diseases and is involved in the antigen presentation modulated by major histocompatibility complex class II molecules, and its overexpression is closely associated with tumor growth, angiogenesis, and metastasis.
The purpose of the present invention is to synthesize new cathepsin inhibitors for the treatment of a disease such as tumor, osteoporosis, arthritis, Ebola virus infection, rheumatoid arthritis, osteoarthritis, an autoimmune disease or a degenerative disease.